Polymer Vesicles - Wiley Online Library

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Encyclopedia of Polymer Sceince and Technology c 2005 John Wiley & Sons, Inc. All rights reserved. Copyright 

POLYMER VESICLES Introduction Vesicles (latin: vesicula = small bubble) are self-supported closed bilayer assemblies of several thousand amphiphiles that enclose an aqueous interior volume. The bilayer is a two-dimensional fluid composed of amphiphiles with their hydrophilic head groups exposed to the aqueous solutions and their hydrophobic tails aggregated to exclude water (Fig. 1). The bilayer structure is highly ordered yet dynamic because of the rapid lateral motion of the amphiphiles within the plane of each half of the bilayer. Cells exploit bilayer structures to create anatomical boundaries, eg in the case of cell membranes which are composed of lipids, proteins, and carbohydrates. During the early 1960s researchers demonstrated that certain classes of lipids, especially phospholipids, could be used to form protein- and carbohydrate-free model membranes. Methods were developed for the preparation of supported bilayer lipid membranes (1), and it was discovered that dried thin films of phospholipids spontaneously hydrate to yield lipid vesicles (2). Vesicles have since then been used as model systems for fluid interfaces and biomembranes (3). Practical applications involving vesicles are in the area of cosmetics and pharmaceutics. The term liposome is frequently interchanged with the term vesicle and is usually reserved for vesicles of glycerophospholipids or other natural lipids. Lipids (greek: lipos = fat) are amphiphiles consisting of two hydrophobic hydrocarbon chains and a polar hydrophilic head group. The most important classes of lipids are phospho- and glycolipids, which contain a phosphate group or a sugar group as head groups. Head groups may in general be ionic, zwitterionic, or dipolar. The typical chemical structures of phospho- and glycolipids are shown in Figure 2. Since Bangham’s 1960s description of lipid vesicles as liposomes (4), many other somes have been created such as virosomes by integrating a cell-binding HIV protein into liposomes (5), niosomes made from nonionic amphiphiles similar in size to lipids (6), polymersomes formed by amphiphilic block copolymers (7), peptosomes made of block copolymers containing polypeptide chains (8,9), and novasomes, multilamellar niosomes developed for topical and cosmetic application (10). In recent years the number of studies on polymer vesicles or polymersomes has strongly increased after first reports by the groups of Eisenberg (11,12), and Bates and Discher (7,13). Since then, amphiphilic block copolymers, rod-coil polymers (14–16), dendrimers (17), and amphiphilic fullerene derivatives (18) have been reported to form vesicular structures. Vesicles can now be made from various polymers, from modified biological to wholly synthetic, from diblock, triblock, 1

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Fig. 1. Lipid and block copolymer vesicles with their typical bilayer structure.

Fig. 2. Chemical structures of a typical phospholipid (phosphatidylcholine), a glycolipid (galactocerebroside), and two vesicle-forming block copolymers, poly(butadiene-b-ethylene oxide) and poly(styrene-b-acrylic acid.) The grey region indicates the hydrophobic interior of the bilayer.

POLYMER VESICLES

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multiblock, and even statistical copolymers in media ranging from complex to neat organics and water (19–21). A summary of vesicle-forming block copolymers is given in Table 1.

Bilayer and Vesicle Formation The formation of vesicles can be viewed as a two-step self-assembly process where first the amphiphile forms a bilayer which in a second step closes to form a vesicle (Fig. 3). In the following we will first discuss conditions that lead to the formation of bilayers and then consider their closure to form vesicles.

Energy and Kinetic Aspects. Thermodynamics of Bilayer Formation. The free energy of a bilayer is mainly determined by the interfacial energy F int of the hydrophobic/hydrophilic interface and the repulsive energy F rep of adjacent amphiphiles (96): F = 2Fint + 2Frep = 2γ a +

2k 2γ = 4γ a0 + (a − a0 )2 a a

(1)

where γ is the interfacial tension, k is a constant describing the repulsion between the hydrophilic part of the amphiphiles, and a is the interfacial area per amphiphile. The factors 2 arise from the contribution of the two monolayers of the bilayer. A further contribution arises from the elastic stretching energy for the molecules to fit into the planar assembly. The minimum free energy can be obtained by setting ∂F/∂a = 0, which yields an equilibrium interfacial area per chain  k a0 = (2) γ which allows to recast the expression for the free energy in equation 1 in terms of γ and a0 . The last term in equation 2 then corresponds to a harmonic potential with an area elasticity modulus γ /a, which will be considered in more detail below. Typical values for the interfacial area per chain are a0 ≈ 0.5 nm2 for phospholipids and a0 ≈ 1–50 nm2 for block copolymers (37,46). Closure to Vesicles. A flat bilayer disk has a rim energy F disk = 2π γ Rdisk (Fig. 3), which can be prevented by bending and closing the bilayer to form a

Fig. 3. Scheme of the formation of bilayers and their closure to form a vesicle. From Ref. 20, with permission from John Wiley & Sons, Inc.

Poly(styrene-b-poly2-β-D-glucopyranosyloxy)ethyl acrylate) Poly(styrene-b-AlaAla) Poly(styrene-b-N,N-dimethylaminoisoprene), PS-PDMAI Poly(butadiene-b-glutamic acid) Poly(styrene-b-phenylquinoline) Poly(isoprene-b-2-cinnamoylethyl methacrylate) Poly(4-aminomethylstyrene-b-styrene) Poly(propylene sulfide-b-ethlyene oxide), PPS-PEO Poly(styrene-b-propyleneimine dendron)

Poly(2-vinylpyridine-b-ethylene oxide), P2VP-PEO Poly(butadiene-b-acrylic acid), PB-PAA Poly(ethylethylene-b-styrenesulfonic acid), PEE-PSSH Poly(butadiene-b-2-vinylpyridine), PB-P2VP Poly(styrene-b-4-vinylpyridine), PS-P4VP

Diblock copolymers Poly(dimethylsiloxane-b-ethylene oxide), PDMS-PEO Poly(methylphenylsiloxane-b-ethylene oxide), PMPS-PEO Poly(styrene-b-ethylene oxide), PS-PEO Poly(styrene-b-acrylic acid), PS-PAA Poly(ethylenepropylene-b-ethylene oxide), PEP-PEO Poly(butadiene-b-ethylene oxide), PB-PEO

Polymer Water Water Water Water, alcohols Water Water Water Water Water Water Water Water CHCl3 Water Water Water Water TFA/CH2 Cl2 THF/hexane Water Water Water

Solvent

Reference

22,23 24 THF, DMF 25–28 DMF, THF, dioxane 11,12,29–45 46,47 THF 48 23,49–54 55 THF 48 56 57–59 DMF 60 HCOOH 61 THF, DMF 62 63,64 THF, DMF, dioxane 65 8,66,67 15,16 68–70 THF, DMF, dioxane 71 72–74 17

Co-solvent

Table 1. Overview of Vesicle-Forming Di- and Triblock Copolymers with Corresponding Solvents and Co-solvents

C60 -(C5 H11 )5 K Poly(dimethylsiloxane-b-2-methyloxazoline), PDMS-PMOX Cyclic poly(styrene-b-isoprene), PS-PI Poly(lactic acid-b-N-isopropylacrylamide), PLA-PNIPAM Poly(lactic acid-b-ethylene oxide), PLA-PEO Poly(caprolactone-b-ethylene oxide), PCL-PEO Poly(3-(trimethylsilyl)propyl methacrylate-b-ethylene oxide), PTMSPMA-PEO Poly(dimethylsiloxane-b-ferrocenylsilane), PDMS-PFS Poly(N-2-(2-(2-methoxyethoxy)ethoxy)acetyl-lysine-b-leucine), PAcOLys-PLeu Poly(ethylene oxide-b-butylene oxide), PEO-PBO Triblock copolymers Poly(styrene-b-methylmethacrylate-b-acrylic acid), PS-PMMA-PAA Poly(acrylic acid-b-styrene-b-4-vinylpyridine), PAA-PS-P4VP Poly(2-methyloxazoline-b-dimethylsiloxane-b-2-methyloxazoline), PMOX-PDMS-PMOX Poly(ethylene oxide-b-propylene oxide-b-ethylene oxide), PEO-PPO-PEO Poly(ethylene oxide-b-propylene sulphide-b-ethylene oxide), PEO-PPS-PEO Poly(dimethylaminoisoprene-b-styrene-b-dimethylaminoisoprene), PDMAI-PS-PDMAI Poly(ethylene oxide-b-dimethylsiloxane-b-2-methyloxazoline), PEO-PDMS-PMeOX Poly(lactic acid-b-PEO-PPO-PEO-b-lactic acid), PLA-PEO-PPO-PEO-PLA

DMF, dioxane, THF 85 DMF/THF 86 87–89 90–92 72 65 75,93,94 95

Water Water Water Water Water

18 75 76 77 78 78 79,80 81 82,83 84

Water Water Water

Water Water Heptane, decane Water Water Water Water methanol Water Water Water

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POLYMER VESICLES

vesicle. The corresponding bending energy is given by F bend = 8π κ (eq. 9), where κ is the bending elastic constant. For a fixed bilayer area A = π R2 disk = 4π R2 ves , a flat bilayer will bend to form vesicles if F bend < F disk , or if the vesicle radius Rves is larger than Rves > Rmin ≈

2κ γ

(3)

The minimal vesicle radius Rmin is increasing with increasing bending modulus and decreasing interfacial tension. Rigid amphiphiles with in-plane order have large bending moduli and therefore form large vesicles. In the case of fluorosurfactants the crystallinity of the bilayers leads to high values of the bending moduli such that rigid bilayer sheets are formed which are unable to bend into vesicles (97). At a given bending modulus an increase of the interfacial tension can induce a transition from planar bilayers to closed vesicles as has been shown for PS-PAA (39). If the interfacial tension is the dominant contribution to the free energy, the bending modulus is κ ∼ γ d2 (eq. 17), and the minimal radius is just dependent on the bilayer thickness Rmin ∼ d2 . Since the bending energy F bend = 8π κ is independent of R, there is no distinguished equilibrium radius for Rves > Rmin . After preparation one mostly obtains polydisperse vesicle samples. The increase in translational entropy will slightly favor the formation of small vesicles. As will be outlined below, a control of the vesicle radius is possible by inducing an asymmetry between the outer and the inner monolayer. Energy Scales. The interfacial energy is in nearly all cases the dominant contribution to the free energy. It is related to the interaction energy ε between the hydrophilic and hydrophobic domains, where typical values are ε ≈ 5–10 kJ/mol (ε ≈ 2 − 4 kB T) per repeat unit, ie CH2 or monomer. ε is related to the FloryHuggins interaction parameter χ = zε/kB T, where z is the coordination number. For a typical value of z = 6 the interaction parameter is χ ≈ 12–24. The interfacial tension is given by γ = ε/a, which for a ≈ 0.5 nm2 has values in the range γ ≈ 17–34 mN/m (γ ≈ 4–8kB T/nm2 ). The key parameter for block copolymer thermodynamics is the segregation parameter χ N, where N = N A + N B is the degree of polymerization of the block copolymer given by the degrees of polymerization of block A and block B, N A and N B , respectively. Throughout the article, “A” will denote the hydrophobic block and “B” the hydrophilic block, respectively. The molar fraction of the hydrophilic block is given by f = N B /N. For typical values of N ≈ 10–1000, the segregation parameter is χ N  100, which corresponds to the limit of strong segregation. The segregation parameter χ N together with the relative block length f dictates the phase behavior of block copolymers, eg the formation of micelles (98), lyotropic phases (99,100) and bulk phases (101,102). A characteristic parameter for amphiphiles is the critical micelle concentration (CMC), which is related to the interaction energy as cCMC ∼ e − Nε/KT . The CMC corresponds to the number of free chains in equilibrium with micelles. For single chain surfactants, typical values are in the range of cCMC ≈ 10 − 2 –10 − 4 M, whereas for double chain lipids typical values are in the range of cCMC ≈ 10 − 9 M. For block copolymers the CMC is expected to be much smaller. Luo and co-workers

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(123) estimated the fraction of free chains to be between 10 − 14 and 10 − 32 M depending on segregation strength. For most vesicle forming systems, the fraction of single free chains is negligibly small. Exchange Kinetics and Freezing. There are two processes that lead to exchange of amphiphiles between micelles or vesicles (103): (1) Exit/insertion of single free amphiphiles: This is the classical mechanism for low molecular weight surfactants; the exchange rate is practically independent of concentration (2) Merger/splitting of two micelles or vesicles upon contact with exchange of amphiphiles: The exchange rate increases with concentration because the number of collisions is increasing The exchange of amphiphiles for both processes is a thermally activated process. As such, the average exchange time (residence time in a micelle or vesicle) is of the order τ ∼ eNε/kT . With increasing interaction energy ε or interfacial tension γ , and as the alkyl or polymer chain becomes longer, the exit rate of a chain becomes appreciably lower. Exchange times for single chain surfactants are τ ≈ 10 − 5 –10 − 3 s, whereas typical values for lipids are τ ≈ 105 s, ie of the order of several hours to days. For polymeric amphiphiles the exchange times are still orders of magnitude larger. The small fraction of single free chains together with slow exchange kinetics essentially freezes the exchange process. A sufficiently fast exchange of amphiphiles is, however, necessary to sustain thermodynamic equilibrium. Therefore vesicles, once formed, are in a metastable, trapped, or quenched thermodynamic state. The number of amphiphiles and therefore their bilayer area is essentially constant on timescales of most experiments. An extreme case is glassy polymers with very slow lateral mobility which can even impede shape changes of polymer vesicles (“frozen” vesicles). The presence of co-solvents can fluidize and activate polymer exchange rates. The influence of solvent composition on vesicle exchange kinetics has been investigated for PS-PAA in DMF/water mixtures (30). When the water content was 7.5 wt%, the morphology of the aggregates depended on the method of sample preparation and the morphological reversibility decreased significantly. Shape transitions such as spheres → cylinders → bilayers were not achievable within a period of several days. The kinetics could be accelerated by increasing the efficiency of adhesive collisions through addition of ions or increase of the polymer concentration.

Packing and Geometry. Packing parameter and Curvature. An important factor determining the shape of self-assembled amphiphilic structures is the size of the hydrophobic part of the amphiphile relative to the hydrophilic part. The shape can be parameterized in terms of the packing parameter , which is given by

=

v Kl2 = 1 − Hlc + c alc 3

(4)

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Fig. 4. Packing of amphiphiles at curved interfaces. The volume v, the surface area a, and the chain length lc determine the interfacial curvature, which is characterized by the two principal radii R1 and R2 .

where v is the volume of the hydrophobic chain, lc is the contour length of the hydrophobic chain, and a is the interfacial area, as shown in Figure 4. The packing parameter is related to the curvature of the hydrophobic/hydrophilic interface as described by its mean curvature H and its Gaussian curvature K (104,105), which are given by the two principal radii of curvature, R1 and R2 H=

  1 1 1 + 2 R 1 R2

(5)

1 K= R1 R2 The simplest shapes are spheres, cylinders, and bilayers which have values of the packing parameter and curvature as shown in Table 2. Conditions for Bilayer Formation. In order to obtain bilayers or vesicles for an amphiphile of length lc , one needs to adjust the interfacial area until the surfactant parameter approaches unity. With the optimum head group area a0 from equation 2 and the definition of the packing parameter (eq. 4), one obtains a simple expression that can serve to rationalize the influence of different parameters on the packing parameter



γ 1/2 v k1/2 lc

(6)

Thus, for fixed length lc , the packing parameter can be increased by (1) increasing the interfacial tension γ , eg by working in aqueous solutions or adding water to organic solvents; Table 2. Values for the Packing Parameter, the Principal Radii of Curvature, Mean and Gaussian Curvature for Different Amphiphile Aggregate Shapes Shape

R1

R2

H

K

Sphere Cylinder Bilayer

1/3 1/2 1

R R ∞

R ∞ ∞

1/R 1/(2R) 0

1/R2 0 0

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9

Table 3. Variation of Different Parameters to Drive Self-Assembly towards Vesicle Formation Parameter Realization

Polymer

Reference

k↓

PS-PAA

11,37,39,44, 50,111,112 113 46,114 46 46 32,39,112 12,115

Decreasing N B

γ ↑ k↓

Addition of water Addition of ions; screens Coulomb interaction

k

pH

k↓

Addition of ionic surfactant (SDS) Core-swelling, screening of PAA

v↑

PS-PEO PB-PEO PEE-PEO PEO-PEE-PEO PS-PAA; dioxane/water PS-PAA PEE-PSSH P4VP-PS; DMF/water; P4VP-PS-PAA; DMF/THF/water PS-PAA; dioxane/water

56 60,86

116

PS-PAA; Dioxane, THF, DMF

38,117

(2) decreasing the chain repulsion, k, eg by decreasing the hydrophilic block length (decreasing hydrophilic fraction f ), screening of electrostatic repulsion via the addition of ions, or screening adjacent chain repulsion by increasing the polymer concentration; (3) increasing the volume, v, eg by swelling with organic solvents. By variation of these factors it is possible to drive amphiphile association in the direction spheres → cylinders → bilayers/vesicles. Detailed theoretical investigations have recently been performed by molecular dynamics simulations (106). Further factors involve temperature and added homopolymer, which affects the stretching energy of the core chains. The variation of these factors and their effects on aggregate morphology have been reviewed (29,32–36,107–110) and are summarized in Table 3. Shape Transitions. The influence of the hydrophilic block length on the micellar shape has been systematically investigated for PS-PAA in DMF/water mixtures (10). As the length of the PAA block decreased, the morphology of the aggregates changed progressively from spheres for PS200 -PAA21 → cylinders for PS200 -PAA15 → bilayers (vesicles and lamellae) for PS200 -PAA8 . It was noted that for block copolymers of similar hydrophobic block length, the values of the interfacial area per chain a0 decrease as the morphology changes from sphere → cylinder → vesicle, which is in accord with the reduced chain repulsion (eq. 2) leading to larger packing parameters for shorter hydrophilic chains (37). As another example, Figure 5 shows the morphologies for a series of PB-PEO block copolymers in aqueous solutions, where a decrease of the hydrophilic block ratio f leads to shape changes from sphere → cylinder → vesicle (102). Similarly,

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POLYMER VESICLES

Fig. 5. TEM (a, b) and optical micrographs (c) of different shapes for a series of PB-PEO block copolymers, ranging from spherical micelles (PB202 -PEO360 , f = 0.64), cylindrical micelles (PB125 -PEO155 , f = 0.55) to vesicles (PB25 -PEO40 , f = 0.38). It illustrates the influence of the hydrophilic block fraction f on the self-assembly structure. From Ref. 20, with permission from John Wiley & Sons, Inc.

an increase of the size of the hydrophobic part at a given hydrophilic block length decreases f , leading to the same series of shape transition as shown for dendronblock copolymers with increasing dendron generation (17). This sequence of shape transitions induced by changes in block length is well known for low molecular weight nonionic surfactants (Cx Ey - series) (118). Phase Diagrams. A phase diagram with the boundaries for the micellar shape transitions as a function of block copolymer composition has been determined for PEO block copolymers (Fig. 6a). The vertical lines are the boundaries separating different micellar shapes. As a rule of thumb, the hydrophilic fraction

Fig. 6. Phase diagrams with shape transition boundaries for block copolymers as a function of (a) hydrophilic fraction f (from Ref. 46, with permission from ACS Publication Division) and (b) water content in dioxane/water solutions (from Ref. 39, with permission from ACS Publication Division). B: bilayers/vesicles; C: cylindrical micelles; S: spherical micelles.

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of the block copolymer should be in a range f ≈ 30–40%, a range which is similar to that for phospholipids, to obtain vesicles (46). The influence of the interfacial tension γ on the micellar shape has been investigated for PS-PAA block copolymers in dioxane/water mixtures. With increasing water content the expected sequence of morphologies, sphere → cylinder → bilayer, is observed (115). Bilayers include vesicles, lamellae, and more complicated structures. The addition of water to dioxane solutions induces aggregation of the polystyrene segments and increases the interfacial tension γ . Figure 6b shows the corresponding phase diagram (39). Not only the water content but also the polymer concentration affects the morphologies and the sizes of the aggregates. It was found that the boundaries of the morphological transitions shift to lower water contents with increasing total block length and with decreasing PAA block length. Further it was found that long core-forming blocks and high water contents favor the formation of vesicles and that short core-forming blocks and low water contents favor the formation of open bilayers. The addition of homopolystyrene changed the morphologies from bilayers or cylinders to spheres (37). Intermediate Shapes. Details of the cylinder → vesicle shape transition have been investigated for PS-PAA block copolymers in dioxane/water mixtures (119). Accordingly, the morphological transition proceeds through a flattening of the rods via a lamellar intermediate state and their closing to form vesicles. The reverse shape transition proceeds via a collapse of the vesicle followed by a rearrangement to form dumbbell aggregates and finally smooth cylinders (40). For amphiphilic block copolymers with compositions f that are intermediate between cylinders and vesicles, Y-junctions and three-dimensional networks leading to a considerable morphological complexity were reported (120). Other complex structures involve the formation of filaments in polyelectrolyte bilayers as a consequence of the balance of long-range repulsive Coulomb interactions and shortrange attractive hydrophobic interactions (59,121,122).

Segregation and Localization. Strong Segregation Limit. So far flexible chain amphiphiles have been considered where a balance of chain repulsion, chain stretching, and interfacial tension together with the hydrophilic/hydrophobic ratio of the amphiphile dictates the aggregate shape. Vesicle formation is generally observed if the hydrophilic fraction of the amphiphiles is in a range f ≈ 30–40%. This range can be much larger if the bilayer structure is further stabilized. This is typically the case for systems with (1) large interfacial energy F int : high energy interfaces in order of increasing interfacial tension are water/oil < water/silicone < water/fluorinated hydrocarbon; (2) small stretching energy F el , which is the case for polymers with small conformational entropy which are typically stiff polymer chains with low internal degrees of freedom or chains with highly localized segments; (3) small repulsive energy F rep , where the repulsion between adjacent polymer chains at the interface is screened, eg by addition of ions or increasing polymer concentrations to high volume fractions.

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Under these conditions the interfacial energy is the most dominant thermodynamic contribution and hydrophobic and hydrophilic domains are highly segregated (strong segregation limit). The system globally minimizes the interfacial area per unit volume, which is given by AV = d

φ l

(7)

where φ is the volume fraction of the hydrophobic domains and d is the dimensionality. Since for planar structures the dimensionality has the lowest value of d = 1, such interfaces are preferred compared to cylindrical (d = 2) or spherical (d = 3) domains. In this regime bilayer structures are preferred irrespective of the shape of the amphiphiles. Secondary Valence Interactions. Under these conditions, the range of stability of planar bilayer structures and vesicles can be quite extended. Low entropy chains include fullerenes, rod-like polymers, and polymers carrying mesogens as well as polymer chains with highly localized chain segments such as branch points in dendrimers (covalent binding) or segments involved in strong functional or complementary interactions including H-bonds, Coulombic, dipolar or π –π interactions, ligand binding, α-helix/β-sheet formation, or other secondary valence interactions. These structures are schematically shown in Figure 7. A well-known case where Coulombic interactions stabilize bilayer structures are mixtures of cationic and anionic surfactants, so-called catanionics (123). A related single component surfactant system which forms equilibrium vesicles over the whole range of volume fractions is single chain sulfonium surfactants (124). Here, the polarizability of the sulfur head group and H-bridges stabilize the bilayer morphology. Vesicle formation was described by Chu and co-workers (18) for pentaphenylfullerene anions, which form small vesicles of 17 nm diameter. Kukula and co-workers described vesicle formation of polybutadiene-b-poly(L)glutamates (7), where the hydrophilic glutamic acid blocks form α-helices. Schlaad and co-workers used polyelectrolyte ionic complex formation of double hydrophilic

Fig. 7. Different amphiphile structures arranged, from left to right, with increasing inplane order, which is an additional driving force for bilayer and vesicle formation. From Ref. 20, with permission from John Wiley & Sons, Inc.

POLYMER VESICLES

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block copolymers to form asymmetric equilibrium vesicles (125). Complexes of block ionomers and surfactants have also been reported to form vesicles (126,127). Even statistical copolymers can form well-defined vesicles by complementary functionalization with diaminopyridine and thymine groups, which are DNA-binding motives (128). Liquid Crystalline Packing. A further factor that stabilizes bilayer structures is a hydrophobic part of the amphiphile, which is shape persistent and has a tendency to form ordered structures. One distinguishes liquid-like structures with short-range order (eg dendrimers, flexible polymer chains) from nematics with additional orientational order (eg rod-like polymers), smectics with additional onedimensional positional order, and crystalline materials with three-dimensional positional order. Increasing in-plane order leads to an increase of the bending modulus of the bilayer resulting in large vesicles. For example, Jenekhe and coworkers (15,16) described giant vesicles that spontaneously formed from rod-coil block copolymers even in organic solvents. The factors leading to bilayer or vesicle formation are schematically summarized in Figure 7, where amphiphiles are arranged according to their in-plane state of order. The pinning of hydrophobic mesogens to an interface is sufficient for the formation of stable bilayer structures. In summary, amphiphiles can be made to assemble into bilayers by adjusting the packing parameter toward unity, for high energy interfaces, or by using amphiphiles with low conformational entropy or using mesogenic amphiphiles. Vesicle Preparation and Size Control. As outlined, the size and shape of vesicles depend to a large extent on preparation conditions. Several procedures are available for the formation of lipid vesicles (129,130), which are in most cases also suitable for the preparation of polymer vesicles. Direct hydration. The simplest procedure is to hydrate a thin film or a solid piece of lipid or block copolymer with an aqueous solution, which leads to the spontaneous formation of vesicles. For film rehydration the amphiphile is dissolved in an organic solvent, which then is evaporated under vacuum or using a nitrogen stream to yield a thin film on a glass or Teflon substrate (131). Upon addition of water, the film swells under formation of a lamellar bilayer phase with vesicles budding or separating at lamellar defects from the film surface (Fig. 8). This method often yields multilamellar vesicles with a broad size distribution. For bulk rehydration stirring, vortexing or sonication is required for a complete hydration of the sample. Electroformation. For electroformation (132), a thin film is deposited on adjacent electrodes (Pt, Au, ITO-coated glass) and the budding process is induced by an alternating current at typically 10 Hz (10 V) (7). The electric field decreases the membrane tension and thereby facilitates vesicle formation. This method yields the largest vesicles (giant vesicles) with diameters up to 100 µm. Hydrating under shear, eg by vigorous stirring, reduces the vesicle size to the submicron range. Details of the shear-induced formation of vesicles from swollen lamellar phases have been investigated (92). Organic Co-solvents. In this method the amphiphile is dissolved in an organic solvent and then mixed with an aqueous solution where it forms vesicles. It is a convenient way for the large-scale preparation of vesicles. Vesicle size and size distribution depends on the details of the mixing process, ie dropwise addition/

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POLYMER VESICLES

Fig. 8. Block copolymer vesicles budding-off from a swollen lamellar phase. After separation, there is no further exchange of block copolymers between vesicles. from Ref. 50, with permission from ACS Publications Division.

injection of the organic solution into water or addition/injection of water into the organic solution both with or without agitation by stirring or sonication. The organic solvent can subsequently be removed by reduced pressure, dialysis, gel filtration, or ultrafiltration. Size Control. Once the vesicles are formed, they will keep their size and size distribution. In order to reduce their size one has to disrupt the vesicle membrane, which is done by strong shear forces, eg by ultrasonic irradiation, ball milling, and high-pressure extrusion through membranes or slits (“French press”). Extrusion through polycarbonate membranes, which are available with pore diameters between 50 and 800 nm, allows a reduction and control in vesicle size and polydispersity to obtain small unilamellar vesicles with size distributions within 10–20%. Fast and direct preparation of small monodisperse vesicles in the size range of 50–200 nm is possible using inkjet printers (133). By appropriate choice of sample preparation method, it is thus possible to tailor the size of the vesicle between 50 nm and 10 µm with narrow size distribution. Glassy Polymers. Vesicle preparation for high molecular weight or glassy block copolymers requires the use of co-solvents. First, the block copolymer is dissolved in a water-miscible common solvent for both blocks (eg dioxane, THF, DMF). Then water is added, which induces the aggregation into micelles. Further addition of water increases the interfacial tension and drives shape transitions to cylindrical micelles, bilayers, and vesicles. Under optimal conditions, it is possible to obtain small, uniform unilamellar vesicles as is shown in Figure 9. In another method, the polymers are dissolved at high temperatures close to their glasstransition temperature. In case of low-boiling solvents, confinement under pressure is required. The formation of vesicles is subsequently induced by cooling (31).

Vesicle Shapes Lipid and polymer vesicles can possess numerous shapes that can be systematically categorized and their energy described by concepts derived from differential geometry.

POLYMER VESICLES

15

Fig. 9. Small, narrow disperse block copolymer vesicles prepared using organic cosolvents. From Ref. 115, with permission from ACS Publications Division.

Curvature Energy. Membrane Energy. Any three-dimensional geometrical object can be characterized by its volume V, surface area A, and its mean and Gaussian curvature H and K, respectively. For bilayer membranes, a further contribution arises from the difference A of the area of the outer and inner monolayer. This leads to five contributions to the free energy (134), which are obtained by integrating over the volume and the surface area of the vesicle as F=F    V + FA + FH+ FK + F A kA kA κ 2 2 ρ¯ dA + (2H) dA + κG KdA + (ρ − dH)2 dA = dV + 2 2 2

(8)

where  is the osmotic pressure, V is the volume of the vesicle, kA is the area elastic modulus, ρ is the deviation from the equilibrium area per chain a0 (eq. 2), κ is the elastic bending modulus, H is the mean curvature (eq. 4), κ G is the Gaussian bending modulus, K is the Gaussian curvature (eq. 4), ρ is the area difference between outer and inner bilayer, and d is the bilayer thickness. Integration of the osmotic pressure  = RT(nin /V–c) over the volume of the vesicle, and of the mean curvature H and area differences ρ and ρ over the area of the vesicle yields (134) RTc kA F= (A − A0 )2 + 8πκ + κG (V − V0 )2 + 2V0 2A

 KdA +

kA ( A − A0 ) 8A

(9)

where c is the concentration of solute outside the vesicle, nin is the moles of solute inside the vesicle, V 0 = nin /c is the volume for which the osmotic pressure vanishes, A0 is the area of the relaxed bilayer, and A and A0 are the actual area difference and the relaxed area difference between the outer and the inner monolayer. Osmotic Pressure. The osmotic contribution to the free energy, F V , arises from the presence of molecules in the solution to which the membrane is impermeable on the timescale of the experiment. This leads to an osmotic pressure  = RT c, where c is the concentration difference between the solutions outside and inside the vesicle. The concentration difference that can be sustained by a curved membrane can be estimated by comparing the osmotic energy to the bending energy F H = 8πκ. For typical values of the bending modulus κ ≈ 1.5 × 10 − 19

16

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J (7,53) one finds at room temperature c ≈ 1 mmol/L. Thus, only minute concentration differences between interior and exterior solution can be balanced by the curvature energy. Large osmotic pressure differences will instead lead to the inward/outward permeation of water and concomitantly an increase/decrease of the vesicle volume until the concentration difference is c = 0 and thus V = V 0 . In practice, this allows one to control the vesicle volume by the molar concentration (“osmolarity”) difference of the inner and outer solutions. Membrane Area. Similar to the osmotic energy, the energy F A involving the total area of the vesicle vastly exceeds the energy of the bending elasticity. Thus the vesicle will adapt an area A = A0 = Na0 that is given by the number N of amphiphiles in the bilayer. The average area of the vesicles is in most cases fixed after sample preparation and may slightly depend on temperature or pH. Genus. As a consequence of the Gauss–Bonnet theorem  KdA = 2π (2 − 2g) (10) the energy due to the Gaussian curvature F K is related to a topological invariant of the vesicle, its genus g. The genus of a surface is given by its number of “holes” or “handles” (see Fig. 10). Therefore, local variations in the Gaussian curvature K have no effect on the global Gaussian curvature as long as the topology is fixed. For any fixed topology or genus, the energy F K is thus constant and can be neglected by setting κ G = 0. Membrane Elasticity. For a vesicle with fixed volume V = V 0 , area A = A0 , and genus the curvature energy of the bilayer membrane reduces to the sum of the remaining two terms of equation 9  κ kA F = FH + F A = (11) (2H − C0 )2 dA + ( A − A0 )2 2 2A which correspond to the two modes of membrane deformation shown in Figure 11. C0 is the spontaneous curvature, which has been introduced to account for a locally preferred curvature of the membrane due to the molecular structure of the amphiphile. Area Difference. A is the area difference between the inner and outer monolayer, which is related to the radius of the vesicle and the bilayer thickness d (see Fig. 12) via

A = Aout − Ain = 8π dR

Fig. 10. Closed surfaces with genus (number of holes) of zero, one, and two.

(12)

POLYMER VESICLES

17

Fig. 11. Stretching (a) and bending (b) of a membrane with the corresponding elastic stretching modulus kA and elastic bending modulus κ. In case of stretching, the stress σ is the force per unit area A = L2 , whereas in the case of bending, the stress is the force per unit cross-sectional area dL. According to simple elasticity theory for thin plates, the two moduli are related by 4κ = kA d2 /12 (135).

The actual area difference will, in general, differ from the optimal area difference

A0

A0 = (Nout − Nin )a0 = Na0

(13)

which is determined by the number of amphiphiles in each monolayer and the equilibrium area per amphiphile a0 . If the interfacial area per amphiphile is the

Fig. 12. Sources of area difference curvature A (insertion, flip-flop) and spontaneous curvature C (block and amphiphile asymmetry, polydispersity) for bilayer structures. The vesicle will adapt a curvature that is a compromise between the two, depending on the related energy.

18

POLYMER VESICLES

same in the outer and inner monolayer, ie aout = ain = a, then the relative area difference A/A is related to the relative difference in the number of amphiphiles

N/N by

A N d = = A N 2R

(14)

with the mean bilayer area A = Na. Thus, for small lipid vesicles with a bilayer thickness d = 3 nm and a radius eg, R = 20 nm, there is a large ( N/N = 30%) difference in the number of lipids in the inner compared to the outer bilayer. This leads to a large free energy contribution related to A, and so small vesicles are often relatively unstable. The area difference A can change by the exchange of lipids between the inner and outer monolayer (“flip-flop”), or the addition (insertion, adsorption, anchoring) of amphiphiles to one monolayer (136) (Fig. 12). Spontaneous Curvature. A different chemical environment on both sides of the membrane (eg different surface charge density, different electrolyte concentrations), an asymmetric molecular shape of the amphiphile, or a different chemical composition of the two monolayers tends to locally curve the membrane (Fig. 12). This membrane-asymmetry-related curvature is described by the spontaneous curvature C0 =

1 R

(15)

of the bilayer. Different chemical environments on both sides of the membrane will in general also lead to differences in the interfacial area per amphiphile aout

= ain and thus in the area difference A. Bilayer asymmetry can already be caused by chain length segregation of polydisperse polymeric amphiphiles between the two monolayers. To reduce steric repulsion, short hydrophilic chains will tend to segregate to the inner monolayer, whereas long chains will preferentially segregate to the outer monolayer as schematically shown in Figure 12. The segregation of block copolymers of different length across the bilayer has been demonstrated by Luo and Eisenberg (41) using pyrene-labeled PS-PAA. Upon addition of a fluorescence quencher (Tl+ ), vesicles prepared with the labeled short hydrophilic blocks experience minimum quenching, since these chains preferably segregate to the inside of the vesicles and are thus largely inaccessible to the quencher. Pyrene residues in the vesicles prepared from the longest labeled hydrophilic chains are 12 times more accessible to the quencher than the pyrene in the vesicles prepared from the shortest labeled hydrophilic chains (Fig. 13). This asymmetry leads to a certain spontaneous curvature C0 . If there is sufficient flip-flop of amphiphiles to adjust the corresponding area difference A, there will be a thermodynamically preferred average radius R ≈ C0 − 1 of the vesicles. As expected, the spontaneous curvature increased, and thus the size of the vesicles decreased with increasing PAA polydispersity, ie increasing bilayer asymmetry (137). This allows to asymmetrically localize two different types of polymer chains if the corona blocks are of sufficiently different length (138). In a further study (42,139), fluorescence quenching

POLYMER VESICLES

19

Fig. 13. Experiments showing preferential quenching of long, fluorescently labeled chains due to their accumulation in the outer monolayer of vesicles. In case of spherical micelles, preferential quenching is neither expected nor observed. From Ref. 41, with permission from the ACS Publications Division.

experiments have shown that this segregation is size dependent. The larger the vesicles, the lower the degree of segregation becomes. The average radius of PS-PAA vesicles could also be changed in solvent mixtures of dioxane/THF/water or DMF/THF/water by changing the water content (11,42,43,111,139). An increase in vesicle size from R ∼ 45 nm to R ∼ 100 nm could be induced by increasing the water content from 25 to 70%, while a decrease in vesicle size could be induced by decreasing the water content by addition of THF/dioxane. The observed change has been attributed to minimization of the interfacial energy, but may also be related to changes in membrane diameter d. The addition of water extracts DMF/THF from the bilayer thereby decreases d, which according to equation 12 leads to an increase of the radius for a given area difference A. The addition of DMF/THF will then lead to the reverse effect. The effect of block lengths, solvent composition, and additives on the vesicle size has been summarized in Reference (45). Equilibrium Curvature. The two sources of curvature ( A, C0 ) correspond to two different radii R. In general, a vesicle will thus be in a stressed state, its radius and shape minimizing the bending energy for a given difference N of the number of amphiphiles in the inner and outer monolayer. The compromise between changes in area difference A and local curvature C0 in equilibrium depends on the relative magnitude of the two bending energies F H and F A . Only if the area difference is A = A0 , so that the corresponding radius R = ( A/8π d) = (1/C0 ), then H = C0 /2 and the corresponding free energy of the membrane (eq. 11) is equal to zero. Under these conditions the vesicle is said to be in its “relaxed” thermodynamic state. An illustrative example for the introduction of spontaneous curvature is bolaamphiphiles. Bolaamphiphiles are membrane-spanning bipolar amphiphiles. They are the most important lipid fraction from archebacteria that exhibit unusual

20

POLYMER VESICLES

stability at extreme conditions of pH and heat. Single chain bolaamphiphiles form extended single-molecule-thick membranes. They are unable to form small vesicles because their vanishing spontaneous curvature requires a large difference

N of lipids in the outer and inner monolayer, which leads to a large free energy contribution related to A. If the spontaneous curvature is increased by addition of cholesterol, small vesicles can be formed by sonication (140). The cholesterol is preferentially incorporated into the outer monolayer of the vesicle bilayer. Bolaamphiphiles with one large and one small head group form asymmetric vesicles with the larger group on the outside surface (141). Chemically Asymmetric Bilayers. Vesicles from the asymmetric triblock copolymer PAA-PS-P4VP have been prepared in DMF/THF/water mixtures (86). Since the ionization of the two hydrophilic P4VP and PAA chains occurs in different pH ranges (for PAA at high pH, P4VP at low pH), a pH-induced flip-flop of the hydrophilic chains could be observed. Vesicles prepared at low pH contained P4VP chains on the outside and PAA chains on the inside membrane to minimize Coulombic repulsion, whereas vesicles prepared from the same triblock at high pH contained PAA outside and P4VP inside. This could be shown by measurements of the ς potential, which changed characteristically when changing the pH of the outside solution. A similar concept has been used for a one-step preparation of block copolymer vesicles with preferentially segregated acidic and basic corona chains (138). Asymmetric PEO-PDMS-PMOX vesicles have been prepared with a preferred orientation of the shorter hydrophilic blocks to the inside of the vesicles (142). The asymmetry was demonstrated by fluorescence quenching experiments similar as in Reference (41).

Vesicle Shapes and Shape Transitions. Phase Diagram. As the surface area A is usually fixed for a vesicle after its preparation, it is useful to consider an area-equivalent radius R = (A/4π )1/2 , which is defined via the radius of a spherical vesicle with the same surface area A. Using the radius R the reduced spontaneous curvature c0 , the reduced volume v, the reduced optimal area difference a0 , and the effective reduced area difference

aˆ 0 are introduced which are given in Table 4. Reduced quantities are convenient measures of vesicles shapes irrespective of vesicle size. The effective spontaneous curvature aˆ 0 is reflecting the different sources of membrane curvature. The constant α = κ/κ gives the relative importance of the two bending moduli with the non-local bending rigidity given by κ = 2kA d2 /π .

Table 4. Parameters Describing the Shape of Vesicles Spherical vesicle Reduced volume Reduced spontaneous curvature Reduced relaxed area difference Effective reduced relaxed area difference

v = 3V/4π R3 c0 = RC0

v=1 c0 = 1

v < 1: deflated vesicle

a0 = A0 /8π dR a0 = 1

aˆ 0 = a0 + (c0 /2π α)

aˆ 0 = 1 + (1/2π α)

aˆ 0 > 1: prolate vesicles

aˆ 0 < 1: oblate vesicles

POLYMER VESICLES

21

Fig. 14. (a) Theoretical phase diagram of vesicle shapes. From Ref. 3, with permission from Elsevier BV. (b) Experimentally observed vesicle shapes of PB-PEO block copolymer vesicles. From Ref. 144. Lipid and block copolymer vesicles show the same structural polymorphism.

For most bilayer systems the constant has values of α ≈ 1. A large area difference

aˆ 0 > 1 tends to curve the membrane outward, whereas a smaller or negative value leads to inward curvature or invaginated vesicle shapes. For a fixed value of α, the two parameters v and aˆ 0 determine the shape of vesicles which can be placed in a two-dimensional phase diagram as shown in Figure 14a (143). There are numerous shapes including spherical, tubular, oblate, and starfish vesicles. Vesicles with outwardly curved shapes and prolate vesicles are in the upper part of the diagram for aˆ 0 > 1, and inwardly curved shapes and oblate vesicles are in the lower part for aˆ 0 > 1. In this phase diagram, all the shapes are drawn to scale, have the same area, and differ only in their values of v and aˆ 0 . Lipid and block copolymers exhibit a similar multitude of shapes as shown for PB-PEO block copolymer vesicles in Figure 14b (144). Polymorphism. As during vesicle preparation the number of amphiphiles in the outer and inner monolayer and thus N is fixed for each vesicle, each vesicle carries its own value of aˆ 0 and thus its own shape. This often leads to a zoo of different vesicle shapes within the same vesicle preparation. From the definition of a0 and equation 12, it follows that

a0 =

¯ N R 2d N

(16)

which means that for a given relative difference N/N between the number of amphiphiles in each monolayer, changes in a0 and thus of vesicle shapes are particularly pronounced for large vesicles (>1 µm) because of the large prefactor

22

POLYMER VESICLES

(R/2d) ≈ 103 . The polymorphism of vesicle shapes is quite commonly observed for large, micron-sized vesicles. Shape Transitions. Shape transitions can be induced by changes in the reduced volume v and the effective spontaneous curvature aˆ 0 and can be mapped as a trajectory in the phase diagram in Figure 14. Changes in v can be induced by osmotic swelling or deswelling of vesicles, eg by changing the concentration (“osmolarity”) of solutes outside the vesicles. Changes in aˆ 0 can occur via changes in area difference A and spontaneous curvature C0 . Changes in temperatures can affect the membrane area and therefore the reduced volume v, but also aˆ 0 which leads to oblique trajectories in the phase diagram. For large reduced volumes, ie quasi-spherical vesicles, there is hardly any membrane area available for shape changes. For v ≤ 0.97 required changes in aˆ 0 for shape transitions are of the order of unity or less, so that shape transitions become achievable. If, on the other hand, by permeation of solvent into the vesicle interior the reduced volume is adjusted to approach unity, all vesicles will become spherical. Common shape transitions include (1) the “pearling” transition, a sequential beading of tubular vesicles; (2) the “budding” transition, where a small vesicle is expelled from the “parent” vesicle; and (3) the vesicle-tubule transition. Pearling Transition. The pearling transition has been investigated for giant tubular PB-PEO block copolymer vesicles. Thermal quenches lead to pearling of the tubes starting symmetrically from the two ends (see Fig. 15) (52). The pearling transition is slow because of the high membrane surface viscosity. The necks close one by one with velocities of the order of a few tens of nanometers per minute, and so complete pearling takes about 2 h. The pearling transition of giant tubular lipid vesicles has also been induced by insertion of O-palmitoyl dextrans into the outside monolayer of the membrane. The insertion leads to an increase in the membrane curvature, which induces the

Fig. 15. Different stages of the pearling transition for a tubular PB-PEO block copolymer vesicle after a thermal quench from 38 to 25◦ C. Compared to lipid vesicles, the transition is extremely slow. The necks close one by one with effective velocities of a few tens of narometers per minute. From Ref. 52, with permission from ACS Publications Division.

POLYMER VESICLES

23

pearling transition (145,146). The concentration critical for the shape transition depends on both the palmitoyl substitution frequency along the dextran and the size of the dextran. Budding Transition. In cells, the budding of vesicles from internal organellae or the plasma membrane is an ubiquitous process and a key process for cellular traffic. In case of giant lipid vesicles, asymmetric insertion of fluorescently labeled PEO with cholesteryl anchor groups into the outer monolayer lead to an increase of the spontaneous curvature and induced budding of starfish vesicles (136). The addition of the cholesteryl anchor increases both components of the effective spontaneous curvature aˆ 0 , the area difference, and the local spontaneous curvature as locally the membrane curves away from the polymer (147). The budding transition is directly related to the fission and fusion process of vesicles. In case of block copolymer vesicles, details of this process have been investigated for PS-PAA using transmission electron microscopy of quenched intermediate states (13,42,139). Accordingly, the fusion proceeds via contact, adhesion, coalescence, and formation of a central, connecting wall. Destabilization of the wall leads to its asymmetric detachment and retraction with final formation of a uniform outer wall. The fission process starts with the elongation of the vesicle, formation of an internal waist, narrowing of the external waist, and final complete separation (Fig. 16). Vesicle fusion and fission kinetics have been followed by turbidity changes after jumps in water content and are found to have relaxation times as fast as seconds. Vesicle-Tubule Transition. Transitions between spherical and tubular vesicles have been reported for a glycopolymer-containing amphiphilic block copolymer by varying the ratio of the co-solvents THF and DMF in aqueous solutions, and by changing the temperature at which the aggregates were prepared (62). Shape transitions involving tubular and starfish vesicles as well as connected tubules have been observed for PS-PEO as a function of water content, polymer concentration, and added ions (25).

Fig. 16. Reconstruction of different stages of the fusion (a) and fission process (b) of block copolymer vesicles. From Ref. 42, with permission from the ACS Publications Division.

24

POLYMER VESICLES

Shape transitions from vesicles to inverted hexagonally packed (hollow) rods were studied for PS-PEO in THF/water mixtures by increasing the water content. The transition proceeds via the formation of hollow regions in the walls of the vesicles, thickening of the walls, and alignment of the rods in a hexagonal pattern leading to the formation of quasihexagonal structures and eventually to the full development of inverted hexagonally packed rods. This mechanism is different from the usually observed fusion of vesicles (25,26). Fascinating intermediate structures such as undulating tubules (44) and ordered toroids (27,148) have been reported for transitions between cylinders and vesicles. High Genus Vesicles. Block copolymer vesicles can also exhibit shapes, which are unknown for lipid vesicles such as stable high genus vesicles (51). Block copolymer vesicles can easily have a genus g > 100, as shown in Figure 17. The local membrane shape indicates that the bilayer has nonzero spontaneous curvature. Experimentally found shapes include vesicles with small and large passages, budded vertices, connected tubes, and spindles, which are stable for different values of the reduced volume and spontaneous curvature aˆ 0 . The range of stability of these shapes can be depicted in a phase diagram similar to Figure 14. The development and stability of holes or pores in bilayer membranes have been studied by poration experiments. These experiments show that pore stability depends strongly on the thickness d of the bilayer (149). For small d, poration leads to large unstable pores and the resulting membrane fragments reassemble into vesicles within minutes. For large d, submicron pores form and are extremely long-lived.

Fig. 17. Comparison of experimental and theoretical shapes for giant high genus PB-PEO vesicles with small passages (a, b), large passages (c, d), and budded vertices (e, f). The scale bar is 10 µm. From Ref. 51, with permission from the APS Associate Publisher.

POLYMER VESICLES

25

Bilayer Properties Physical properties of the vesicle membranes such as elasticity, toughness, stability, and permeability determine to a large extent the performance of vesicles in applications. These properties are largely determined by the interfacial tension and membrane thickness, the latter being adjustable via the chain length of the membrane forming block copolymer. This allows one to tailor membrane characteristics within a range outlined in the following sections. Mechanical Properties. The mechanical properties of bilayers such as their bending elasticity (Fig. 11) can be understood by a rather simple model. If the bilayer is assumed to be a thin, solid-like film, there is a simple relation between the area elasticity modulus kA and the bending modulus κ (19,135,150) (Fig. 11), ie κ=

1 kA d2 48

(17)

where d is the bilayer thickness. If the interfacial tension is the dominant contribution to the elastic properties of the membrane, then a comparison of the coefficients of the area elastic energy terms in equations 2 and 9 implies (96) kA = 4γ

(18)

Thus, both the bending modulus and area elasticity are directly related and are determined by the interfacial tension. Thickness. The thickness of polymersome bilayers is several times greater than that of typical phospholipid bilayers in natural membranes. Lipid bilayers have a hydrophobic core thickness that is in a very narrow range of d ≈ 3–4 nm to be compatible with integral membrane proteins. For self-assembled bilayers of PEE-PEO vesicles, the hydrophobic core thickness increases with increasing molecular weight from d ≈ 8–21 nm (see Fig. 18) to more than 100 nm (53,151– 153). The observed d ∼ M 0.5 scaling is typical for random coil polymers and agrees with molecular dynamics simulations of block copolymer bilayers (106). Elasticity. The elastic behavior of membranes has been studied using micropipette techniques on giant vesicles (>5 µm). These techniques allow one to simultaneously measure membrane stretching and bending moduli. The experiments show an interface-dominated elasticity. Membrane stretching moduli are found to be independent of chain length (Fig. 19a). A value of kA ≈ 120 mN/m is reported for PEE-PEO vesicles, which is in good agreement with γ = kA /4 ≈ 30 mN/m consistent with an oil/water interface (eq. 18). Bending and stretching elastic constants are in a range of values typical for lipid membranes (53). The well-investigated unsaturated lipid DPPC (dipalmitoyl phosphatidylcholine) mixed with cholesterol (1:1) has a value of kA = 102 mN/m corresponding to γ ≈ 25 mN/m (155). For polymersomes, the bending elastic constant κ is reported to be ∼35 kT (7), which is in good agreement with equation 17. Data confirm the expected square dependence of the bending modulus on membrane thickness (Fig. 19b) (154).

26

POLYMER VESICLES

Fig. 18. Increase of the membrane thickness d with the hydrophobic molecular weight for PEE-PEO vesicles. Data (triangles) are also shown for membranes of various phospholipids. Block copolymer vesicle bilayers can be more than an order of magnitude thicker compared to phospholipid vesicles. From Ref. 151, with permission from the ACS Publications Division.

Toughness. Membrane stability as defined by the maximal area strain α c = (Ac – A0 )/A0 increases with increasing molecular weight, approaching a universal limit set by the interfacial tension. The sustainable critical strain is α c ≈ 0.19, which considerably exceeds the range of α c = 0.03–0.06 typical of natural lipid membranes (Fig. 20); lipid membranes cannot be strained without rupture, osmotically or otherwise, by more than about 5% in surface area. Polymersomes are almost an order of magnitude tougher and sustain far greater area strain before rupture compared to lipids. From the data, a critical yield stress of σc = kAdαc ≈ 1 MPa can be estimated. One expects that both critical yield tensions and strains increase linearly with the bilayer thickness. Nonlinear responses and memory effects emerge with increasing molecular weight, indicating the onset of chain entanglements at higher molecular weight (151). Dynamics. For polymersomes, measurements of the lateral diffusivity (47) and the membrane viscosity (152) indicate that membrane fluidity decreases strongly with increasing molecular weight. The decreases are most drastic when the chains are long enough to entangle. Fluorescence recovery after photobleaching has been used to determine lateral diffusion in block copolymer bilayers (Fig. 21). The experiments yield a time constant τ from which the lateral diffusion coefficient D = l2 /2τ is calculated. In the fluid Lα phase, lipid molecules have lateral diffusion coefficients of

POLYMER VESICLES

27

Fig. 19. (a) Area elasticity modulus kA and (b) bending modulus κ as a function of the bilayer thickness d. The experiments are in agreement with equations 17 and 18. From Refs. 151 and 154, with permission from the ACS Publications Division.

28

POLYMER VESICLES

Fig. 20. Maximum area strain for lipid and block copolymer bilayers as function of molecular weight. This experiment demonstrates the increased toughness of block copolymer vesicles compared to lipid vesicles. From Ref. 151, with permission from the ACS Publications Division.

Fig. 21. Fluorescence recovery after photobleaching for a polymersome tip pulled into a glass micropipette. The recovery of the tip intensity can be monitored by fluorescence imaging showing the lateral diffusivity in the bilayer. From Ref. 47, with permission from the ACS Publications Division).

POLYMER VESICLES

29

D ≈ 10 − 7 –10 − 8 cm2 /s. Depending on molecular weight, block copolymers have much lower values of D ≈ 10 − 9 –10 − 11 cm2 /s for molecular weights between 4000 and 10,000 g/mol (47). The surface shear viscosity of block copolymer vesicles is about 500 times higher than those found in common phospholipid bilayers (53). The experiments involve pulling out a tether from an immobilized polymersome and following its relaxation back to the vesicle body. This provides an estimate of the viscous coupling between the two monolayers composing the polymer membrane. For giant vesicles of approx. 100 µm diameter, typical relaxation times for thin tethers of 50 µm length and 0.1 µm diameter are of the order of 30–90 s. The detected intermonolayer friction is about an order of magnitude higher than for phospholipid membranes. Stability. Because of their increased toughness and bending elastisticity, block copolymer vesicles are more stable compared to lipid vesicles. Block copolymer vesicles have been reported to be stable over several years with no changes in size or size distribution (156). Electromechanical Stability. The electromechanical stability of diblock copolymer membranes is several times larger compared to lipid membranes (152). At zero mechanical tension, the breakdown potential V c for polymersomes with a membrane thickness of d = 15 µm is V c = 9 V, compared to 1 V for liposomes with d = 3 nm. The breakdown potential increases with membrane thickness up to a limit set by the interfacial tension (53,152). The electromechanical stresses at breakdown universally exhibit a Vc2 dependence. The membrane capacitance shows the expected strong d dependence. Thermal Stability. The thermal expansion coefficient for lipid membranes far from phase transitions is generally in the range α T = 0.001–0.01 K − 1 (157), which is similar for polymersomes with values of α T ≈ 0.002 K − 1 (49), albeit with no thermal transitions. The thermal stability of vesicles is of importance, if autoclaving is needed for sterilizing vesicles that are too large for filtration through 0.22 µm filters. When polymersomes are autoclaved in dilute suspension for 15 min at a maximum cycle temperature of 121◦ C (2 atm), about 10% of the vesicles have retained their contents (sucrose solution). Interestingly, the distribution of vesicle sizes shifts to smaller and tighter distribution. It is speculated that because of area expansion, small vesicles have budded off (49). Stability to Lysis. The addition of surfactants eventually leads to lysis of vesicles. In general, the stability of polymer vesicles against lysis is expected to be higher compared to lipid vesicles, because of the reduced entropy of mixing of polymer and surfactant chains which leads to a reduced partition of surfactants in the polymer membrane. When using Triton-X as a nonionic surfactant, it was found that the concentration of surfactant required for dissolution after a fixed amount of time increases nearly linearly with membrane thickness (158). Because of low partition of hydrocarbon surfactants, vesicles formed by siloxane and fluorinated polymers are expected to have relatively high stability. The incorporation of PEO-PPO-PEO (Pluronic L31) surfactants into preformed giant PEE-PEO vesicles has been investigated and was found to be weak and reversible (159). The incorporation reduced the area expansion modulus kA by almost a factor of 2, while dramatically increasing the vesicles susceptibility to lysis. Additionally, water permeability was increased by a factor

30

POLYMER VESICLES

of 2. Surfactant incorporation rates were proportional to the free surfactant concentration.

Permeability. Permeation Coefficient. Vesicles will slowly release encapsulated molecules by permeation through the vesicle bilayer. The release rate is determined by the permeation coefficient P (in m2 /s), which is related to the solubility or the partition coefficient S of the compound between the aqueous phase and the bilayer, and to the rate of diffusion or diffusivity D of the compound in the bilayer, ie P = SD

(19)

In a fluid membrane, diffusion coefficients of small molecules have the same magnitude as in bulk solvents. Therefore, the factor determining the permeability is the partition coefficient. Small ions whose low solubility in hydrophobic environment leads to very small values of the partition coefficient exhibit very low permeabilities. The permeability of small polar solutes across a bilayer or membrane can be greatly enhanced by introducing specific carriers. A small solute X can be transported by a carrier C such that the complex CX has a much higher partition coefficient compared to the bare solute X. Common ion carriers, or ionophores, are valinomycin for K+ and crown ethers for alkali ions. Solutes can also cross the membrane through channel proteins, eg gramicidin. If the permeation occurs via Fickian diffusion driven by the concentration gradient across the bilayer, the permeation time τ is related to the permeation coefficient as τ=

Rd Vd = 3P AP

(20)

where R is the radius, V the volume, A the surface area of the vesicles, and d is the bilayer thickness. Smaller vesicles exhibit a faster release because of the larger surface/volume ratio compared to larger vesicles. Often, the permeation of molecules is compared in terms of their permeability p = Pd (in m/s), since most lipid bilayers have a similar bilayer thickness of d ≈ 3 nm. For practical applications, equation 20 allows one to tailor release rates τ − 1 via bilayer thickness and vesicle radius, which themselves can be controlled via polymer block length and vesicle preparation method. Because of the larger thickness of polymer bilayers compared to lipid bilayers, the permeability of molecules can be adjusted to be much slower. Permeation Experiments. Encapsulated fluorescent and radiochemical labels are commonly used to evaluate permeabilities. Three different types of experiments allow to determine the permeation coefficient. (1) In one experiment the release of labeled molecules into the exterior solution is monitored in a closed cell. If the initial concentration of the label inside the vesicle is c0 , and in the exterior solution is equal to zero, and if the volume encapsulated by the vesicles is much smaller than the volume of

POLYMER VESICLES

31

the sample cell, V ves  V cell , then the concentration of the labeled molecule in the exterior solution is increasing with time t as c(t) = c∞ (1 − e − t/τ )

(21)

where c∞ = V ves c0 /V cell is the saturation concentration in the exterior solution for t → ∞. For short times (t  τ ), c(t) = c∞ t/τ , which allows to obtain the permeation time and thus the permeation coefficient from the initial linear increase of the concentration with time. The concentration c∞ can be obtained after lysing the vesicles via addition of a surfactant, where all labeled molecules are released into the exterior solution. Conveniently, one monitors the increase of the fluorescence intensity of an encapsulated self-quenched dye, eg 50 mM 6-carboxyfluorescein (6-CF) (160) or calcein (161), as it permeates the vesicles. [3 H]Glucose is more generally applicable because it does not interact with charged lipid membrane surfaces and can be used at low ionic strength. Alternatively, a dilute solution of 6-CF (10 µm) is encapsulated and Co2+ (500 µm) is added to the external solution which quenches the fluorescence. Permeation of either Co2+ ions or the dye across the membranes causes a reduction in fluorescence intensity. (2) In the second type of experiment the vesicle with encapsulated solute is placed in an ultrafiltration cell which is continuously flushed with solvent at a rate ν 1 (in mL/min) (162,163) c1 (t) =

Vves c0 e − t/τ v1 τ

(22)

The permeation coefficient can be obtained from the exponential decay, provided that the elution rate is faster than the permeation rate of the vesicles, v1  V ves /τ . This method does not rely on fluorescence labels and can be used for any solute. In addition, this method more closely resembles the in vivo situation where vesicles release solutes that are instantaneously carried away by the blood stream. (3) In a third type of experiment the vesicles are investigated under equilibrium with free exchange between encapsulated and nonencapsulated solute. The exchange rate and fraction of encapsulated solute can be determined with pulsed field gradient nuclear magnetic resonance (PFG-NMR). Using this technique, the permeability of polyethylene oxide PEO through P2VPPEO vesicle bilayers has been determined as a function of molecular weight of PEO (55). The analysis yielded a nearly linear dependence of the logarithmic transmembrane exchange rate on the hydrodynamic radius of the polyethylene glycol (PEG) molecules. The method does not require to separate encapsulated from nonencapsulated molecules. It allows to estimate the encapsulated molar fraction of PEO chains, which was in the range of 2%.

Barrier Properties. The water permeability p of lipid bilayers is reported to be in a range of p = (5–100) × 10 − 6 m/s. In comparison, polar solutes such as

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glucose, glycerol, and urea have much smaller values of p = (5–300) × 10 − 10 m/s. Small ions such as Na+ , K+ , and Cl − have the lowest permeabilities, in the range of p = (1–100) × 10 − 14 m/s (164). Even though the bilayers are only 3 nm thick, they provide an effective barrier to ions and other polar compounds. The permeability of polymersome membranes for a given solute has been reported to be at least 10 times smaller compared to common phospholipid membranes (8). Retention of encapsulants (eg dextrans, sucrose, physiological saline) over periods of months has been observed for small ∼100 nm polymersomes prepared by liposome-type extrusion techniques (49) as well as with ∼10 µm giant vesicles. Permeability and membrane hydration have been shown to be inversely related to the bending modulus (165). It is suggested that membrane permeability and membrane hydration are related to the ability of the membrane to locally bend so as to create holes for solute permeation.

Cross-Linking, Anchoring, and Templating of Membranes A major problem of lipid vesicles in pharmaceutical applications has always been their minor stability. Stabilization of vesicles can be achieved by introduction of covalent cross-links or the attachment of stabilizing polymers (87,166–168). The most common methods involve the (1) formation of vesicles with polymerizable amphiphiles and subsequent polymerization (166,167,169); (2) solubilization or association of polymerizable monomers to the amphiphile or vesicle bilayer with subsequent polymerization; (3) association or anchoring of polymers for vesicle bilayer stabilization. The polymerization of lipid assemblies was first demonstrated in monolayers, vesicles, and extended bilayers and subsequently in cast multilayers, black lipid membranes, and tubules. During the 1970s several groups demonstrated that fatty acids with an incorporated reactive group, eg photopolymerizable diacetylenes, could be polymerized in a monolayer (170,171). The introduction of synthetic double-tail amphiphiles, coupled with the successful demonstration of polymerization of fatty acid monolayers, led directly to the next step in the early 1980s. Vesicles of a polymerizable double-chain ammonium salt have been described with a methacrylate at the end of one hydrocarbon chain (1) (Fig. 22) (172). In short order three additional groups (173–175) reported the synthesis and polymerization of lipid diacetylenes (2, 3) in bilayer membranes. Within a year, the synthesis and polymerization of dienoyl lipids (4) (176,177), methyacroyl lipids and other diacetylenes (178), and styryl lipids (179) were reported. Polymerizable Amphiphiles. Polymerization of amphiphiles with only one reactive group per lipid such as 1–3 yield vesicles that contain several polymer chains, termed polymerized vesicles. The presence of a second polymerizable group per molecule (4) allows cross-linking of the polymer chains and thus crosslinking of the bilayer structure. Such reactive groups include diacetylene, acryloyl, methacryloyl, itaconyl, dienoyl, muconyl, styryl, vinyl, thiol, and isocyanates. The

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Fig. 22. Chemical structure of polymerizable and cross-linkable lipids.

use of carotene side chains for the preparation of cross-linked vesicles has also been demonstrated (180). Polymerization can be induced by photo, thermal, or redox initiation. In most cases, the vesicle size and shape are not significantly changed by the polymerization reaction. Cross-Linking. Cross-linking increases membrane stability to detergents and organic solvents. The polymerization of monosubstituted lipids 1–3 in vesicles leads to moderate changes in stability, whereas more significant changes are

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found only after polymerization of bis-substituted lipids. For example, polymerized vesicles from mono-methacryloyl lipids may still be disrupted by detergents and organic solvents, albeit at somewhat higher concentrations than required for unpolymerized vesicles. In contrast, polymerized vesicles from the bisdienoyl PC (4) are quite stable to detergents and organic solvents (181). The polymerization of 4 yields vesicles that are stable enough to maintain their spherical shape in the vacuum chamber of a scanning electron microscope (182). Polybisdienoyl PC vesicles are not solubilized by 2 mM sodium dodecyl sulfate (183). Measurements of the release rate of encapsulated [3 H]glucose through vesicles before and after polymerization show that the permeabilities of vesicles of mono-methacryloyl lipids are reduced upon complete polymerization to 30–50% of the unpolymerized control vesicles (181). A much larger reduction (2 orders of magnitude) in membrane permeability is observed on photopolymerization of the bis-methacryloyl lipid 4, which allows cross-linking of the vesicle bilayer. Thus the formation of linear chain polymers in vesicles does not significantly alter the physical properties, whereas polymerization of disubstituted lipids can lead to crosslinked polymer networks with a corresponding large change in vesicle properties. Polymerizable Counterions. Ion exchange of the counterions of ionic lipids vs polymerizable counterions such as choline methacrylate presents an additional route for vesicle stabilization much like the formation of the actin network of natural membranes. Photopolymerization yields vesicles with surface-associated polyelectrolytes. These vesicles were reported to have a similarly reduced permeability as polymerized mono-methacryloyl lipid vesicles (184). Polymerizable Block Copolymers. Unsaturated hydrocarbon polymers with a large number of polymerizable double bonds can be used to prepare fully cross-linked vesicle membranes. This has been demonstrated for polybutadiene block copolymers (23,168) and methacroyl-functionalized block copolymers (87) using free-radical polymerization, and for polycinnamoylethylmethacrylate block copolymers using a [2+2] photoaddition (68). Poly(ethylene oxide-b-3(trimethoxysilyl)propyl methacrylate) vesicles could be efficiently cross-linked with triethylamine (79,80). PLA-PNIPAM block copolymer vesicles were crosslinked by chain extension of the PNIPAM block using hexamethylene diacrylate (77). In case of the polybutadiene vesicles, the membrane transformation from liquid to solid state is directly observable upon osmotic deflation of the originally spherical vesicle as creases, folds, wrinkles, and dents proliferate like the deflation of a micron-sized rubber ball (Fig. 23). The cross-linked vesicles do not dissolve in chloroform, a common solvent for both blocks. Encapsulated sucrose was not lost in chloroform, indicating membrane stability on a molecular scale. The results imply defect-free membranes many microns-squared in area. Surface elastic moduli as well as sustainable wall stresses are orders of magnitude greater than any natural lipid membrane. Cross-linked vesicles could be dried, stored at room temperature for days, and then rehydrated to their original average diameter and volume (87). Mixing of saturated (PEE) and unsaturated (PB) block copolymers allows to tune the bilayer elastic constants as well as the rupture strength over a broad range (168). Preblending with a non-cross-linkable diblock copolymer of PEO-polylactic acid undermines vesicle stability in chloroform/water solutions (185). The results prove miscibility and stable integration of a binary block

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Fig. 23. Deflation of a non-cross-linked (left) and a cross-linked polymersome (right). The latter deflates with dents and wrinkles, characteristic of a solid-like membrane. From Ref. 168, with permission from the ACS Publications Division.

copolymer mixture in a membrane, and allow to realize a first-order scheme for controlled release of encapsulants. Solubilization of Monomers. Hydrophobic monomers such as methacrylates (186) and styrene (187) can be solubilized into the bilayers of lipid vesicles. These monomers can subsequently be polymerized within the membranes by an UV-induced free-radical polymerization, which leads to the formation of a quasi-two-dimensional polymer entanglement network. The polymerized vesicles are stable to addition of surfactant (bare liposomes are not), can be isolated by lyophilization, and subsequently redispersed in aqueous media by sonification. Depending on the dimensions of the templating vesicles, polymer hollow spheres can be produced with diameters ranging from several tens of nanometers up to hundreds of micrometers (188–191). After extraction of the surfactant matrix, the polymer particles contract considerably without loss of their spherical shape. By the same method, water-soluble polyelectrolyte nanocapsules have been prepared (192–194). These particles show a reversible pH- and ionic strength-dependent swelling transition, causing a considerable increase (decrease) of their radius together with a change of the permeability of the polyelectrolyte walls, which can be used to trigger the release of encapsulated materials. Adsorption of Polyelectrolytes. Some water-soluble polymers adsorb to neutral or charged membrane surfaces by hydrophilic or ionic interactions which can stabilize or destabilize vesicles. Polyelectrolytes such as polyionene-6,6 (195,196) and poly(L-lysine) (197,198) have been adsorbed to negatively charged lipid vesicles and effects on bilayer thickness and polymer conformation have been investigated. Studies of the adsorption of polyanions to positively charged lipid vesicles are of importance for gene transfection of plasmid-DNA in gene therapy. The adsorption of poly(α-ethylacrylic acid) (PEAA) has been used to make lipid vesicles pH-sensitive. Acidification induces conformational changes of PEAA, leading to disruption of the vesicles (199,200). Anchoring of Polymers. Synthetic polymers with hydrophobic side groups interact with membranes by insertion of these so-called anchors into the bilayer. Polysaccharides such as pullulan and amylopectin, which were derivatized with palmitoyl groups, are sufficiently hydrophobic to insert into lipid bilayers (201). If a sufficient amount of polysaccharide is added after vesicle formation, the

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vesicles are coated only on the exterior surface. The bound polysaccharide reduces the vesicle permeability to water-soluble markers and decreases the sensitivity of the vesicle lipids to enzymatic hydrolysis by phospholipases (201). The surface characteristics of such polysaccharide vesicles are sufficiently different from those of uncoated vesicles in that they display an unusual distribution of vesicles in the body. The lung uptake of vesicle-encapsulated label is five times greater for Opalmitoyl amylopectin-coated vesicles than for uncoated vesicles. Monoclonal antibody fragments attached to polysaccharide-coated vesicles bind to specific cells with a higher frequency than those attached to antibody-free coated vesicles (202). Pluronic-type PEO-PPO-PEO triblock copolymers were anchored into bilayers of soybean lecithin to prepare sterically stabilized vesicles (203–205). Vesicles with anchored polymer are reported to have an increased stability with respect to cation (Na+ , Mn2+ )-induced flocculation. Also the addition of other hydrophobically modified polymers to lipid vesicles lead to a stabilization of the vesicles (206). Templating. Solutions of multilamellar block copolymer vesicles could be used as templates for the preparation of vesicular structured monolithic silica using a sol/gel process (57). This method can be used to freeze vesicular structure for analysis, and also for the preparation of mesoporous silica with slit pores. Thin films of templated multi-bilayer vesicular structures of PS-PEO could also be prepared using sol/gel chemistry (28). The films were prepared from dilute PS-PEO/THF/water/silica precursor mixtures via solvent evaporation. For the silica/diblock copolymer films with regular mesophases, copolymer removal produced also mesoporous structures.

Biomedical Applications Vesicles are intensely investigated with respect to their use as transportation systems for pharmaceutical and cosmetic applications (207,208). The potential of lipid vesicles is rooted in the notion that natural lipids will be at least partially biocompatible (209). Via conjugation with biological ligands different biomedical functions can be implemented into the vesicle (Fig. 24), including (1) actuator functions for temperature-, pH-, salt-, and light-triggered release; (2) the incorporation of adhesion sites, eg RGD sequence, sialyl-Lewisx , HIVTAT sequence; (3) the conjugation of recognition sites, eg antibodies like anti-PECAM to target lung endothelia or anti-CD3 to peripheral blood cells (210); (4) a “stealth” layer to prevent unspecific recognition; and (5) integral proteins that provide channel and transport functions. Conjugation can be achieved by covalently attaching a ligand to the end group of the peripheral polymer block, eg via activated ester synthesis, or by reconstitution of a protein or ligand into the vesicle bilayer. By using a mixture of functionalized and unfunctionalized block copolymers, vesicles with optimal ligand spacing can be prepared for efficient binding to cell surfaces.

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Fig. 24. Biomedical functions that can be implemented into vesicles. From Ref. 20, with permission from John Wiley & Sons, Inc.

Problems can consist in the chemical and colloidal stability, eg upon oral administration, and in the low permeation through cellular membranes. For this reason vesicles are mostly administered parenterally, ie intravenously, subcutaneously, or intramuscularly. Their tissue specificity is first confined to the cells of the reticuloendothelial system which recognizes them as foreign microparticles and transports them to liver and spleen. Their biodistribution can be regulated by surface modification, eg by placing PEO (stealth systems) or monoclonal antibodies (site-specific targeting) to the vesicle surface. By this means their residence time and bioavailablity can be increased (211). Biocompatibility. The biocompatibility of materials is influenced predominantly by the material surface. Materials with hydrophilic surfaces are often wellsuited, because hydrophilic, noninteracting surfaces are difficult to recognize by living systems and have low cytotoxicity. Stealth Layers. It is known that PEO (or PEG, which is chemically the same) enhances the biocompatibility of polymeric materials. PEO is water soluble, chemically stable under physiological conditions, and shows low unspecific binding of proteins. Liposomes containing lipid-conjugated PEO as a hydrophilic layer do not interact with and are difficult to be recognized by living systems and have been called “stealth” liposomes. Blood plasma proteins, which generally adsorb to artificial surfaces and mediate clearance by immune system cells, are sterically delayed in deposition into or onto “stealth” membranes. After intravenous injection stealth liposomes are cleared more slowly from the blood circulation than are conventional liposomes. PEO chains can prolong circulation half-lives from a few hours to tens of hours. The extended circulation time allows stealth vesicles loaded with anticancer drugs (eg doxorubicin) to be distributed to distant and well-hidden

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tumors (212). Eventually, liposomes are engulfed by phagocytic cells of the liver and spleen. For optimal stealth properties, liposomes would have to be densely covered by a PEO layer. However, PEO liposomes are limited in their ability to integrate high molar ratios of PEO lipid because of shape transitions to micellar structure as a result of the increasing interfacial curvature and lower packing parameter. Polymersomes have the advantage that vesicles are entirely composed of PEO-based block copolymer amphiphiles and are not limited by PEO-driven micellization. Blood Plasma Stability. Polymer vesicles are found to be stable for at least 5 days in plasma when suspended at room temperature and kept well-mixed (49). Under quiescent conditions vesicles are stable for almost 60 h. At longer times, aggregation of settled-out vesicles is observed. Within the aggregates, no vesicle fusion is observed because of the steric stabilization of the PEO chains. Phagocyte Stability. Of particular importance is the assessment of the interaction of vesicles with the cellular components of blood, particularly granulocytes, which are the predominant circulating phagocytes. Neither adhesion nor stimulation of phagocytes are apparent when giant polymersomes are held in direct contact. When placed in contact with a white cell (Fig. 25b), vesicles do not exhibit any adhesion or other cellular response for up to 30 min, despite the presence of 20% plasma. In comparison, 1–2 min of contact between a yeast particle and a white cell (neutrophil leukocyte) leads to strong adhesion (49). Proliferating cells are unaffected when cultured for an extended time with an excess of polymersomes. Similar in vitro tests with macrophages, endothelial cells, and myoblasts further indicate the inertness of the polymersome surface. The dense PEO layer prevents the deposition of phagocytic ligands such as plasma C3b on the vesicle surface, thereby repelling phagocyte adhesion. The lack of polymersome recognition by phagocytes is important for prolonged circulation upon intravital injection. In Vivo Performance. Polymersomes are found to have in vivo circulation times of τ 1/2 ≈ 18 h, which is about twofold longer than PEGylated or stealth

Fig. 25. Exposure of a yeast cell (a) and a polymersome (b) to a white cell. The yeast cell is rapidly engulfed, whereas there is no response upon contact with the polymersome. From Ref. 49, with permission from John Wiley & Sons, Inc.

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Fig. 26. Fluorescence images (a) and number of polymersomes (b) after different circulation times in rats. The experiment shows the delayed clearance of polymersomes from the blood stream. From Ref. 213, with permission from Elsevier B.V.

liposomes. Fluorescently labeled vesicles were injected into rat tail veins with subsequent bleeds taken to monitor vesicle clearance from the circulation (213) by taking a fluorescence image of centrifuged platelet-poor plasma (Fig. 26). From image analysis, the decay of the number of vesicles with time is obtained, which is fitted to an exponential to derive the circulation half time τ 1/2 . As with lipid vesicles, the circulation time is limited by uptake into the liver and spleen. In vitro incubations of polymersomes in plasma indicate plasma protein adsorption during circulation, which leads to engulfment of the vesicles by phagocytes. Toxicity. Toxicity is found to be a minor problem and many vesicle systems have passed standard toxicity tests. The hydrophilic PEO block effectively screens the influence of the hydrophobic cores on timescales of the measured circulation half-life τ 1/2 . With respect to long-time applications, PEE is structurally similar to low-density polyethylene, which is commonly used in implants and is generally considered bioinert (214). PB is also understood to be bioinert as it is commonly chosen for the hydrophobic block in triblock copolymers (215,216). In a more prolonged model test of biocompatibility, nondifferentiated C2 C12 cells were incubated with polymersomes. The cells remained well spread with no evidence of cytotoxicity. As shown by the Trypan blue exclusion assay, polymersomes had no significant effect on cell survival.

Encapsulation and Release. Encapsulation Methods. Vesicles can solubilize and encapsulate hydrophilic as well as hydrophobic compounds. Hydrophobic compounds (eg carotene, vitamin E, Taxol, CdTe-quantum dots (20)) are solubilized into the hydrophobic interior of the bilayer. Solubilization can be achieved by stirring the

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Fig. 27. Methods for encapsulation and release of hydrophilic drugs using vesicles. From Ref. 20, with permission from John Wiley & Sons, Inc.

hydrophobe in the aqueous vesicle solution or by dissolving the vesicle-forming amphiphile and the hydrophobe in an organic co-solvent with subsequent transfer into water. Because of the small mixing entropy, polymer vesicles have a higher selectivity for the solubilization of hydrophic compounds compared to lipid vesicles. Hydrophilic drugs can be encapsulated by dissolving the vesicle-forming amphiphile in the aqueous drug solution (Fig. 27). Removal and recycling of nonencapsulated molecules is possible by ultrafiltration, gel filtration, dialysis, or centrifugation. Polymer vesicles can retain encapsulated molecules over periods of days to weeks. Encapsulated substances include fluorescein (7,102), rhodamin (68), eosin, myoglobin, hemoglobin (7), BSA (7), dextrans (7), PEG (55), sucrose (7), physiological saline, doxorubicin (78,217), β-lactamase (218), and cylindrical micelles (219). As has been shown, encapsulation of enzymes into liposomes or polymer nanocapsules protects them against proteases and denaturation (218). Particularly interesting is the integration of channel proteins into bilayers, which is discussed below. Sustained Release. Depending on permeation coefficient, vesicle radius, and bilayer thickness, encapsulated low molecular weight solutes will be released on timescales of minutes to days. This can be used for the controlled release of drugs, where the dose can be predicted from the encapsulated volume and the initial drug concentration in the vesicle using equation 21. Large ionic solutes, in particular proteins, will have low release rates and are practically permanently encapsulated until the vesicle is ruptured. Triggered Release. By triggering solution conditions vesicles can be spontaneously dissolved with a concomitant release of the encapsulated substance. This can be achieved with vesicles containing polymer blocks with solubilities responding to changes in pH, temperature, and ionic strength. Other stimuli include UV, light, enzymes, and reducing agents which degrade the polymer block. pH-Triggered Release. The pH-induced release is of relevance for the delivery of drugs and genes into the cytosol via endolysosomal acidification and

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Fig. 28. Shrinking and lysis of P2VP-PEO vesicles by addition of dilute acetic acid. The arrows indicate the formation of holes in the membrane. From Ref. 224.

escape for applications in chemo- and gene therapy. The acid-catalyzed and thus pH-dependent hydrolysis of plasmenylcholine vesicles has been used to release encapsulated calcein (220). With the pH decreasing from 6.3 to 2.3, the time to release 50% of the calcein decreased by 3 orders of magnitude from 50 h down to 3 min. The acid-catalyzed hydrolysis of the vinylether linkage of PEG-DOPC lipids has been used to trigger the release of calcein from lipid vesicles at pH